Project Summary Many patients with hematological disorders rely on hematopoietic stem cell (HSC) transplants for treatment. However, for numerous patients, this option is dramatically impacted by the lack of optimally matched donors. Generation of HSCs ex vivo would greatly improve treatment for these patients. Understanding how HSCs form in vivo is crucial for the efficient derivation of HSCs ex vivo. During development, HSCs originate from a subset of endothelium, known as hemogenic endothelium. HSCs form through a process known as endothelial-to- hematopoietic transition (EHT). The complete genetic and molecular mechanism underlying EHT is still not fully understood, but it is known that RUNX1 is absolutely necessary for EHT. Ectopic expression of RUNX1 in embryonic non-hemogenic endothelium at E8.5 is able to induce EHT and blood cell formation. However, four days later in mouse development, RUNX1 is not as effective at reprogramming fetal endothelium to undergo EHT. Efficient hemogenic specification of fetal endothelium requires twice as much RUNX1, and proceeds via a different route than that of embryonic ECs. We hypothesize that the differential hemogenic competency observed between fetal and embryonic endothelium is due, at least in part, to changes in chromatin organization that prevent RUNX1 from binding key target genes. In order to understand how RUNX1 can efficiently specify embryonic endothelium as hemogenic, we will identify direct targets of RUNX1 and determine if these targets are marked by repressive heterochromatin in fetal endothelium. Additionally, we will determine if there are underlying baseline differences in chromatin structure between fetal and embryonic endothelium by profiling the genes in lamina associated domains (LADs) and using stochastic optical reconstruction microscopy (STORM) to determine global chromatin and nucleosome organization. Additionally, we will use DNA fluorescent in situ hybridization (FISH) to determine if important RUNX1 targets are localized to the nuclear periphery in fetal endothelium, which is what is preventing these genes from getting activated. These experiments will provide insight on the precise chromatin landscape and gene expression necessary for efficient hemogenic specification from endothelium. These studies will provide potential avenues for ex vivo generation of HSCs for therapeutic uses.